Adjuvant compositions and medicinal mixtures comprising them

ABSTRACT

Acrylic acid polymer adjuvant compositions comprising a mixture of an acrylic acid polymer crosslinked with a polyallylsaccharide and a physiologically acceptable electrolyte have properties which greatly enhance the ease with which they may be formulated with active agents in the production of therapeutic mixtures of the adjuvant composition and active agent. Solutions of the therapeutic mixture have viscosities which are lowered to the point where they can be utilized commercially in a practical manner, while at the same time retaining the favorable adjuvant effects.

United States, Patent Lund [ Mar. 4, 1975 [75] Inventor: Lloyd J. Lund,Moraga, Calif.

[73] Assignee: Cutter Laboratories, Inc., Berkeley,

Calif.

22 Filed: Dec. 22, 1972 21 Appl. No.: 317,752

UNITED STATES PATENTS 4/l965 Lund 424/89 9/1969 Hardy 424/89 3,639,5772/1972 Urton et al. 424/92 Primary Examiner-Shep K. Rose Attorney,Agent, or FirnzMillen, Raptes & White 57 ABSTRACT Acrylic acid polymeradjuvant compositions compris ing a mixture of an acrylic acid polymercrosslinked with a polyallylsaccharide and a physiologically acceptableelectrolyte have properties which greatly on hance the ease with whichthey may be formulated with active agents in the production oftherapeutic mixtures of the adjuvant composition and active agent.Solutions of the therapeutic mixture have viscosities which are loweredto the point where they can be utilized commercially in a practicalmanner, while at the same time retaining the: favorable a'djuvanteffects,

6 Claims, No Drawings 51 ADJUVANT COMPOSITIONS AND MEDICINAL MIXTURESCOMPRISING THEM BACKGROUND OF THE INVENTION This invention relates toadjuvants, more specifically to adjuvant compositions comprising anacrylic acid polymer cross-linked with polyallylsucrose orpolyallylpentaerythritol and to medicinal compositions comprising suchadjuvants.

It is well established that the therapeutic effect of medicinal agentswhen administered subcutaneously or intramuscularly can be prolonged orenhanced by incorporating adjuvants with the medicinal agent. Forexample, antibody titers can be significantly enhanced in a host when anantigen is injected which has associated with it a substance whichdelays the release and distribution of the antigen throughout thetissues of the host. Substances such as mineral oil and aluminumhydroxide produce this adjuvant effect. They serve several functions;namely, they adsorb the medicinal agent, as for example, an antigenicsubstance, and prevent rapid distribution of the antigen intosurrounding tissues. Being relatively insoluble and non-metabolizable,they keep the antigen more or less localized at the site of injection sothat the antigen is not quickly dispersed, thus giving the lymphocytesof the host an opportunity to generate antibodies. They also act as anirritant which promotes the routing of lymphocytes to the irritated sitethus bringing the antibody producers in contact with the antigen. Suchadjuvants, however, are not too satisfactory since they generallypromote the formation of sterile abcesses and mineral oils may beimplicated in the formation of tumors.

In US. Pat. Re. No. 26,963, reconstituted collagen is disclosed as anon-toxic adjuvant. It is stated that the collagen in the activeagentcollagen combination, after injection, gels to a solid mass andretains the active agent in the injection site, a condition which favorsretardation of the release of the active agent and hence allows theeffect of the active agent to be maintained for a prolonged period oftime. Furthermore, the observation is made that, at a given pH, thecollagen may be dispersed in direct proportion to the amount of sodiumchloride added to it. The life of this adjuvant, hence its period ofeffectiveness, is decreased by the inclusion of sodium chloride.

The use of a polymer of acrylic acid cross-linked with various polyolcompounds as an adjuvant is described in US. Pat. No. 3,178,350. Suchpolymers are commercially available under the trademark Carbopol.Carbopol 934, for example, is the polymer where the cross-linking agentis polyallylsucrose.Carbopol 94l is a lower molecular weight member ofthe class of Carbopols. These polymers form aqueous gels whose viscosityincreases with increased pH or increased concentration of the Carbopol.According to the disclosure in US. Pat. No. 3,178,350, mixtures ofCarbopol 934 or Carbopol 941 with various virus vaccines at aboutneutral pH, resulted in greatly enhanced antibody production in the hostwhen the Carbopols were in the range of 0.25 to 0.50 per cent of themixture. Unneutralized Carbopol was considerably less effective anadjuvant, probably because the antigen is destroyed at the low pH of theacidic Carbopol. It is belived that Carbopol behaves in a manner similarto other gels such as collagen and aluminum hydroxide; namely, theactive agent is adsorbed on the polymer and the combination is retainedat the injection site. The polymer is only slowly dispersed and theactive agent is retained at the site for prolonged slow release.

Carbopol in its neutralized states, however, forms very viscoussolutions or gels in the range of 0.25 to 0.50 per cent after mixingwith an active agent and is still too viscous to draw into and dispensefrom a hypodermic syringe with ease. Furthermore, the viscous nature ofthe material at these concentrations makes it very difficult to blend ormix uniformly with an active agent which is in a dry state or in anaqueous solution, particularly in production scale.

Theexcellent adjuvant properties of the Carbopol cross-linked acrylicacid polymers would be most desirable if the high viscosity of usefulranges did not render it impracticable in formulation and dispensingoperations.

It is a primary objective of this invention to provide effectiveadjuvant solutions of certain acrylic acid polymers while at the sametime controlling the viscosity thereof to permit them to be readilymixed with an active agent and easily injected into a host.

A further object is to provide concentrated solutions of such polymerswhich may be easily mixed with solutions of active agents to giveeffective adjuvant mix tures without materially lowering theconcentration of the active agent.

SUMMARY OF THE INVENTION The compositions of this invention are adjuvantmixtures of a substantially neutral injectable acrylic acid polymercross-linked with polyallylsucrose or polyallylpentaerythritol capableof exerting an adjuvant effect in vertebrates in admixture with anamount ofa physiologically acceptable electrolyte effective to lowersubstantially the viscosity of aqueous solutions of the polymers, andantigen-adjuvant biologics comprising them.

DETAILED DISCUSSION The viscosity of Carbopol type cross-linked acrylicacid polymer solutions can be reduced in accordance to this invention toa level which allows the solutions to be readily mixed with solid orsolution forms of biologically active agents and allows the mixture tobe easily dispensed through a hypodermic needle, by admixing anelectrolyte with the polymer. It is known that an electrolyte, such assodium chloride, increases the dis persibility of a, collagen adjuvant.However, this increased dispersion, resulting from a decrease inviscosity, leads to a decrease in the adjuvant effect. It is also knownthat electrolytes will decrease the viscosity of Carbopol gels orsolutions. On the basis of the detrimental effect that sodium chloridehas on collagen, it would be expected that Carbopol would likewisebecome less effective as an adjuvant in the presence of an electrolyte.Therefore, it was of considerable surprise to discover that even thougha solution of Carbopol whose viscosity hadbeen greatly lowered by theinclusion of an electrolyte, this solution could be mixed with an activeagent, an antigen for example, and the adjuvant effect of the polymer isretained.

The adjuvant of this invention comprises an aqueous solution of apolymer as defined herein and a physiologically acceptable electrolyte,in a molar weight proportion of about 1 of the polymer (assuming a minimum molecular weight of about 200,000) to about 800 to 8,000 of theelectrolyte. The adjuvant solution should preferably be about neutral,that is, in the pH range of 6.0-8.0 to avoid destruction of activeagents which may be mixed with the adjuvant solution. Many activeagents, antigens for example, are degraded in acidic or basic media.

The polymers employed as adjuvants include the class of colloidallywater-soluble acrylic acid polymers cross-linked with polyhydroxycompounds in which the hydrogen atoms of at least some of the hydroxygroups have been replaced by unsaturated aliphatic groups, e.g.,polymers of acrylic acid cross-linked with from 0.75 to 2.00 percent ofpolyallysucrose and polyallylpentaerythritol, as described in U.S. Pat.No. 3,178,350. Typical of these compounds are those made by the B. F.Goodrich Chemical Company and designated by the trademark Carbopol.Carbopol934 is the acrylic acid polymer cross-linked with approximately1 percent polyallylsucrose. Carbopol-934 is the preferred adjuvantcompound although other Carbopols such as Carbopol- 940 and Carbopol94lare also acceptable. The concentration of the polymer in the adjuvantsolution can vary from about 0.10 per cent to about 3 per cent or more.The higher concentrated solutions are of particular advantage for mixingwith solutions of an active agent, for example bacterins, which cannotbe diluted appreciably without lowering the potency of these bacterinsto levels unacceptable to the U.S. government regulatory agencies.Higher concentrations of the polymer can be diluted by mixing with avolume of a solution of an active agent which brings the concentrationofpolymer in the resulting adjuvant-active agent mixture to a lowerlevel which is sufficient to produce an adjuvant effect. The finalconcentrations of the polymer in the mixture with the active agentshould be at least 0.10 percent and can be as high as about 1.0 percent,preferably in the range of about 0.20 to about 0.50 percent. If theactive agent is in dry form, it is merely dissolved or suspended in thepolymer solution of a concentration known to give good adjuvant effects,usually about 0.2 to 0.5 percent or more.

Solutions of the polymer in a pH range of 6-8 are very viscous even at aconcentration of around 0.25 percent and at about 0.5 percent or higherthey form gels. It becomes almost physically impossible to mix an activeagent into gels containing 0.5 percent or higher of polymer and achievea homogeneous mixture, particularly on a production scale. Mixtures ofan active agent and the polymer in which the concentration of thepolymer is about 0.25 to 0.50 percent are generally too viscous to drawinto or expel easily through a needle of a hypodermic syringe.

These drawbacks for using an otherwise effective polymer adjuvant areovercome by the incorporation with the polymer an amount of electrolytesufficient to reduce the viscosity of the polymer solution to usefullevels, e.g., about 500 to 50,000 cps, preferably about 500 to about40,000 cps. The amount of electrolyte which should be added willgenerally be dictated by the initial viscosity of the polymer at theintended concentration in the active agent-adjuvant solution. The amountof electrolyte generally should not greatly exceed isotonicity in thefinal adjuvant-active agent mixture. Usually lesser quantities will berequired to bring the viscosity of the mixture down to a useful level.Any physiologically acceptable electrolyte may be used, as

long as it is physiologically acceptable and thus not detrimental to thehost. Examples are alkali-metal, alkaline earth and ammonium salts ofmineral and organic acids, e.g., sodium chloride and bromide; potassiumchloride and bromide; ammonium, magnesium and calcium chloride; sodium,potassium, ammonium and magnesium sulfate; sodium and potassium mono-,di-, and tribasic phosphates; sodium and potassium acetates, sodium andpotassium propionates; sodium and potassium succinates; sodium andpotassium maleates; sodium and potassium tartrates; sodium and potassiumlactates; ammonium, sodium and potassium citrates; sodium, potassium andmagnesium gluconates; and sodium ascorbate. Of these, sodium phosphates,sodium chloride, sodium citrate and magnesium sulfate are preferred. Insome instances the active agent itself may be ionic and will effect alowering of viscosity. Nevertheless, sufficient electrolyte should beincorporated in the polymer adjuvant solution to lower its viscosity toa level which will allow the solution to be mixed relatively easily withthe active agent.

The viscosity of the polymer-electrolyte adjuvant solution, prior tomixing with the active agent, regardless of whether the active agent isin the dry form or in a solution or suspension, should not be greaterthan about 50,000 cps as measured in the following manner: Approximately400 ml. of the adjuvant solution is placed in a 600 ml. beaker and a No.7 spindle is placed into the solution and rotated at 10 rpm whilerecording the reading on a Brookfield viscometer, model RVT at 25Ci0.5C.To achieve a viscosity of about 50,000 cps or lower, for ease in mixingthe adjuvant solutions with solutions or suspensions of an active agent,particularly when higher polymer concentrations are involved, anelectrolyte is mixed with the polymer or aqueous solution thereof in anamount which will lower the viscosity of the adjuvant solution to 50,000cps or less, prefera' bly about 500-40,000 cps, more preferably about500-32,000cps. For ease in passing through hypodermic needles, the finalviscosity of the solution of the mixture adjuvant and active agent,should generally be about several hundred to a few thousand centipoise.

ln U.S. Pat. No. 3,178,350, there is disclosed an adjuvant solutioncontaining only Carbopol. Good adjuvant properties were obtained usingCarbopol at 0.25 and 0.50 percent. The viscosities of these solutions atpH 7 were 12,000 and 60,000 cps, respectively using a No. 7 spindle anda speed of 10 rpm. Although it required some effort, particularly withthe 0.5 percent solution, they were finally mixed with dried influenzavaccine, for example, and the mixtures could be made to pass through a20 gauge needle but only with difficulty. For example, with the 0.25percent solution, it requires approximately 40 seconds to draw 5 ml.into a syringe and about 15 seconds to expel the 5 ml. The 0.5 percentgel could not be drawn through the needle. lf 5 ml. was placed into asyringe, it required about 20 seconds and considerable pressure to expelthe contents through the needle. After adding sodium phosphates tosimilar solutions in amounts to make them about half isotonic, theviscosities were 400 cps and 600 cps, respectively. These solutionscould be readily mixed with dried vaccine and made to pass through 20gauge needles with ease and rapidity. The adjuvant effect did notdiminish, even though the viscosity had been greatly decreased. The termactive agent as used herebefore is meant to include therapeutic agentsof many types whose effeet may be prolonged or enhanced by theirinclusion with the adjuvant polymers. Among these are antigenicsubstances which stimulate the production of antibodies and includeviruses such as influenza (monovalent or polyvalent, human or equine),measles, mumps, poliomyelitis, canine distemper, encephalomyelitis,rabies, foot mouth disease, bovine diahrrea, infectious bovinerhinotracheaitis and Newcastle disease viruses; toxoids, such as thoseof Clostridium perfringens (Types B, C, and D), Clostridium tetani, anddiphtheria; bacterins, such as those of Clostridium chauvoei,Clostridium septicum, Clostridium hemolyricum, Clostridium novyi,Clostridium sordellii, Leptospira, streptococcus, staphlococcus,pneumococcus, pseudomonas, H. pertussis, fowl cholera, and erysipelas;rickettsia, such as rocky mountain spotted fever, typhus, Q fever;allergens, such as pollens, dust, danders or extract of same; andvenoms, such as bee sting and snake venoms; and the like.

The adjuvant solutions may be prepared by several procedures. In onemethod, the electrolyte and polymer are intimately blended in the drystate, then added gradually to water, preferably warmed to about 50 to70C., while stirring at about l2000 rpm. Following dissolution of themixture, the pH is adjusted to the desired value, somewhere in the rangeof 6-8, preferably about 6.5-7.5, by the addition of an aqueous solutionof a base such as sodium hydroxide or ammonium hydroxide. Additionalwater is added to bring the polymer to the desired final concentrationand the solution may then be sterilized by conventional means. Theadjuvant solution may also be prepared by blending an electrolyte with apolymer which has been neutralized with a base such as sodium orammonium hydroxide and isolated in a solid state. The blend is readilydissolved in the appropriate amount of water. The preblending 0f thepolymer and electrolyte greatly facilitates the dissolution of thepolymer since high viscosities are not encountered as would be the casewhen polymer is first dissolved and then the electrolyte is added.

Solutions or suspensions of the adjuvant solution in combination with anactive agent may then be prepared. In those cases where the active agentis added as a solid, the concentration of the polymer in the adjuvantsolution will generally be in a lower range, most usually about 0.2 to0.5 percent, so that the final concentration of the polymer in theadjuvant-active agent mixture will be the same as in the adjuvantsolution. When the adjuvant solution is to be mixed with an active agentwhich is in the form of a solution or suspension, such as solublebacterin or virus vaccine solutions, then the concentration of thepolymer in the adjuvant solution will usually be much greater,generally, in the range of about 0.5 to 3 percent or higher, so that theresulting mixture of one volume of the adjuvant solution with one to tenor more volumes of vaccine solution, for example, will contain theadjuvant polymer at a lower but still very effective level. In effect,the potency of the vaccines are not materially reduced by this dilutionwith the polymer adjuvant solution. This is very important when one isdealing with vaccines whose potencies are borderline and which would beimpossible or commercially impracticable or detrimental to concentrateby means such as ultrafiltration or dialysis. An alternative to mixingvery concentrated solutions of polymer adjuvant with vaccines withborderline potencies is to dissolve a blend of neutralized polymer andelectrolyte directly into the vaccine solution. Generally, however, itis more convenient and reliable in large production batches to mix thepolymer as a solution with the vaccine solution.

Although not necessary or essential for the adjuvant effects of thepolymer in the polymer-electrolyte solution, other materials may beincluded. For example, various physiologically acceptable emulsifiersmaybe added to the water to speed up the dissolution of the polymer whenpreparing the adjuvant solution. The emulsifier may be a non-ioniccompound, such as Tween (polyoxyethylene sorbitan monooleate), Span 20(sorbitan monolaurate) or others of this type, alone or in combinationsor mixed with vegetable oils. The emulsifier, when used, generally doesnot exceed about 0.05 percent in the final adjuvant-active agentmixture. The inclusion of buffers is sometimes desirable for maintainingpH stability of the adjuvant solution. Alkali metal phosphates, such asmonosodium and disodium phosphates are preferable. They not only serveas a buffer but being electrolytes also contribute to the lowering ofviscosity of the polymer solution. Phosphates may be used in combinationwith other electrolytes such as sodium chloride, the phosphate acting asa buffer and both effecting a lowering of viscosity. Non-toxic materialssuch as gelatin and collagen, may also be incorporated into the adjuvantsolution. Their presence appears to provide a more elegant appearance tothe solution. Although concentration may vary, a preferred concentrationappears to be about 1% in the adjuvant-active agent mixture. Preferredadjuvant-active agent compositions of this invention are those adaptedfor intramuscular (l.M.), intraperitoneal (I.P.) and/or subcutaneous(SQ) administration.

The invention thus relates to improved adjuvant solutions and toinjectable mixtures thereof containing biologics. The adjuvant solutionscomprise about 0.10 to about 3.0 percent of a substantially neutral,colloidally water-soluble acrylic acid polymer cross-linked with apolyalkylsucrose or polyallylpentaerythritol and a physiologicallyacceptable electrolyte. The electrolyte is present in an amount whichwill at least lower the viscosity of the adjuvant solution within therange of about 500 to about 50,000 cps depending upon the relativeconcentration of the polymer. The electrolyte concentration may be ashigh as that which produces isotonicity in the final injectable solutionof the mixture of the adjuvant solution and the biologic. The injectablecomposition resulting from mixing the adjuvant solution and the biologicshould contain between 0.1 and 1.0 percent of the polymer, preferablyabout 0.2 to 0.5 percent. The injectable composition should contain anamount of electrolyte relative to the polymer concentration whichproduces a viscosity from about 200 to about l0,000 cps, preferablyabout 200 to 2,000 cps. Thus, for example, a solution containing 0.5percent of the polymer would have a viscosity of about 60,000 cps andwould be too viscous to mix conveniently with a biologic solution. Ifsodium phosphate is added to this solution to give a concentration of1.4 percent (about isotonic), the viscosity of the resulting solutiondrops to about 600 cps and this solution can be readily mixed with abiologic. A 50:50 mixture of this adjuvant solution and a biologicsolution would produce an injectable composition containing 0.25 percentof the polymer and 0.7 percent of the phosphate electrolyte (aboutone-half isotonic) and having a viscosity of about 400 cps. If, forexample, the adjuvant solution contains 2.5 percent of the polymer andno electrolyte, the viscosity is about 166,000 cps and the adjuvantequal volume of a 2.2 percent A1(OH) suspension.

C. Same as A. but replacing the phosphate buffer with an equal volume ofthe Carbopol-sodium phos phate electrolyte adjuvant solution of Example5.

23 t: be mixedkwith a biologi? g additiqn 5 D. Dried human influenzavaccine (PR8) mixed p P ate to e a concelltmtlon o percent m with theCarbopol solution of Example 2 which conthe ad uvant solution, theviscosity drops to about t amed no electrolyte 48,000. This solution hasa low enough viscosity that it can be mixed with a solution of abiologic. Mixing nine Test Three groups of 24 SWISS Webster i werepal-ts of a biologic Solution with one part of this adjw l0 Inoculatedsubcutaneously with 0.5 ml. of either vacvant solution results in afinal concentration of 0.25 P preparatlon or Fourteen days later 12percent of the polymer and 0.7 percent of the phosfrom f l group w e theSera were Pooled phate. The viscosity of the final mixture is about 400and the remamrrrg 12 l In each group were Elven cps, well below What isacceptable for ease in injecting second 0.5 ml. inoculation. Pooled serafrom the secand adjuvanted biologic Solution 15 0nd group of 12 werecollected after 14 days. A fourth In order to more clearly disclose thenature of the group of mlee were given y one dose of present invention,specific examples illustrating typical the vaeeme Prepararrerr; Serumwas e9lleered frm compositions of the adjuvant solutions and combinalrrlree on the 1 y and on the 28th y l tion with specific active agentare hereinafter derrters e e r e r Pooled Sera y the Standard scribed 20hemaglutmatron inhibition test (HAI).

ADJUVANT SOLUTIONS OF CARBOPOL Test 2. A group of 8 rats was inoculatedsubcutane- All weights are expressed in grams of Carbopol or ously with0.5 ml. of the D vaccine preparation, serum electrolyte in 100 ml. ofsolution. pH was adjusted with was collected on the 14th day, andanalyzed by the HA1 20% NaOH. test.

Viscosity at 25 1 05C. Carbopol" Scale 1-100 Example 9341 Electrolyte pHSpindle RPM Reading x Factor cps l 0.25 none 6.8 7 3 4000 12,000 2 0.5none 6.8 7 10 4000 60,000 3 2.5 none 6.65 7 10 41.5 4000 166,000 4 0.250.47 Na HPO 6.9 7 100 1 400 400 0.23 NaH- PO .H O 5 0.5 0.94 Na HPQ, 6.97 10 1.5 400 600 0.46 NaH,PO .H O 6 2.5 4.7 Na HPO 6.95 7 10 12 400048,000

2.3 NaH,P0,.H O 7 2.5 9.4 Na HPO 7.0 7 l0 8 4000 32,000

4.6 NaH PO .H O 8 2.5 2.5 NaCl 7.0 7 10 10 4000 40,000 9 2.5 5.0 NaCl6.60 7 10 4.5 4000 18,000 10 2.5 1.2 MgSO 7.0 7 l0 8 4000 32,000 11 2.55.0 Na citrate 6.5 7 10 13.5 4000 54,000

(dihydrate) 12 2.5 10.0 Na Citrate 6.7 7 10 6.5 4000 26,000

' (dihydrate) Carbopol 940 13 2.5 5.0 NaCl 6.9 7 10 10 4000 40,000

Carbopol 941 14 2.5 2.5 NaCl 6.8 7 10 7.5 4000 30,000

"1 stands for pharmaceutical grade. "'Solution also contains equal partsTween 80 and Span (total 0.1 g.), 1 ml. cottonseed oil and 2.5 g.gelatin.

MIXTURES OF CARBOPOL ADJUVANT WITH HA] Tm l ACTIVE AGENTS Days PostAgainst Vaccine Preparation Inoculation Strain Strain Example 15 A A2Influenza Virus r r A (Control no ad uvant) 14 128 2 (2 inoculations) 28128 16 The following vaccine preparations were made:

B (A1(OH),-, adjuvant) 14 128 2 A. Bivalent equine influenza vaccinesolution con- (Zlnoculatrons) 28 256 I6 taining formalinkilled Strain A,(A/Equine 1/Pa./63 st- C (Carbopol adjuvant ram) and Strain A (A/equme2/Miam1/63 strain) with electrolyte) 14 256 2 mixed with an equal volumeof 1.4 percent sodium (zmeculauers) 64 phosphate buffer (controlvaccine, no adjuvant pres- S512 ent). 5

C (Carbo I ad'uvant B. Same as A. but replacing phosphate buffer with anwith elgimlyle) I5 256 32 -Continued HAl Titer Days Post Against VaccinePreparation Inoculation Strain Strain (1 inoculation) 28 512 128 D(Carbopol adjuvant Strain A no electrolyte) i4 256 l inoculation) Theseresults show the adjuvant effect of Carbopol to be superior to that ofAl(Ol-l) with respect to influenza virus vaccines and demonstrate thatthe adjuvant effect of Carbopol is not diminished even though theviscosity of the Carbopol vaccine mixture has been decreased by theinclusion of the phosphate electrolyte.

Example 16 Equine Influenza Virus Horses were inoculated intramuscularlywith 1.0 ml. of a 50-50 mixture of the bivalent equine influenza(Strains A and A vaccine solution'and the Carbopolphosphate electrolyteadjuvant of Example 5. Serum samples were obtained on days 0, 21, 41 and63. All horses showed slight to moderate pre-existing antibody levelsdue to previous infections with the viruses. Earlier studies on twogroups of 13 horses had demonstrated little or no antibody response toeither strain could be elicited up to 63 days following injection of thesame virus in phosphate buffer and in Al(Ol-l) Days Post HAl TitersAgainst Horse Inoculation Strain A Strain A 6 These results from testingon another specles further Example 17 Clostridium chauvoei. BacterinStrains Nos. 5686, 5677, 5677-2, and 5987 of C. chauvoei were grown inbeef broth and killed with formalin. Equal parts of the four wholecultures were combined for the vaccine. A portion was centrifuged, thesupernatant fluid was reserved, and the paste of whole cells was washedand resuspended in isotonic phosphate saline buffer to the originalvolume before centrifugation. The following mixtures were then prepared:

A. 87 parts whole culture 13 parts of 2.2 percent Al(OH) suspension.

B. 87 parts supernatant fluid l3 parts of 2.2 percent Al(OH) suspension.

C. 87 parts resuspended cells 13 parts of 2.2 percent Al(OH) suspension.D. 90 parts whole culture 10 parts of the Carbopolphosphate electrolyteadjuvant of Example 6.

E. 90 parts supernatant fluid 10 parts of the Carbopol-phosphateelectrolyte adjuvant of Example 6 F. 90 parts resuspended cells 10 partsof the Carbopol-phosphate electrolyte adjuvant of Example 6.

Mixture Survival 8/10 7/10 6/10 10/10 l0/l0 l0/l0 0/10 Controls (nobacterin) C. chauvoei vaccines containing no adjuvant seldom will passpotency test requirements since potencies of production lots which havenot been concentrated by some means are generally borderline. Adjuvantsare therefore most desirable for such vaccine preparations. The aboveexperiment demonstrates how it is possible to take advantage of the goodadjuvant properties of Carbopol without significantly lowering thepotency per ml. of the bacterin. This is accomplished by adding only avery small volume of a very concentrated solution of the Carbopol to thebacterin solution. Mixing the bacterin solution with the concentratedCarbopol solution is possible only by virtue of the greatly reducedviscosity of the Carbopol solution resulting from the inclusion of theelectrolyte. Without the electrolyte, the 2.5 percent Carbopol solutionwould exist as a stiff gel which could not possibly be mixed with thebacterin so lution without a great deal of difficulty, require aprolonged period of time and render the operation commerciallyimpracticable.

Example 18 Clostridium sordellii Toxoid 7 Ninety parts of this fluid wasmixed with 10 parts of the IQ Carbopol-phosphate electrolyte adjuvant ofExample 6. Ten mice were injected subcutaneously with 0.5 ml. of themixture on days 0 and 14 and challenged on the 28th day with C.sordellii spore.

Mixture Survival C. sordellii toxoid Carbopol 8/10 Example 19Clostridium septicum with C. chauvoei Bacterin Various mixtures of C.chauvoei and C. septicum were prepared as follows:

A. 2 parts C. chauvoei whole culture with 1 part C.

septicum whole culture.

B. 1 part each of the two whole cultures plus the paste fromcentrifugation of 1 part chauvoei whole culture.

C. Paste from 1 part chauvoei and 1 part septicum whole culture.

Bacterin-Adjuvant Mixture Challenged with Survival A C. chauvoei spore8/l0 C. septicum spore 5/5 B C. chauvoei spore 9/10 C. septicum spore5/5 C C. chauvoei spore 6/10 C. septicum spore 5/5 D C. cliavuuei spore8/9 C septicum spore 5/5 Controls C chauvoei spore 0/l0 (no bacterin) C.spelicum spore 0/5 Good adjuvant effects were demonstrated for Carbopol934P with respect to C. septicum. Results from Examples 18 and 19further show the advantages of being able to mix concentrated Carbopolsolutions (because of the viscosity lowering effect of the electrolyte)with two other Clostridium bacterins whose potencies are normallyborderline and obtain good adjuvant effects. Without adjuvant of anykind, vaccine preparations of C. sordellii or C. septicum seldom passpotency test requirements.

What is claimed is:

1. A biologic solution comprising an antigen which stimulates theproduction of antibodies and an adjuvant therefore consistingessentially of a colloidally watersoluble neutralized polymer of acrylicacid cross-linked with from 0.75 to 2.00 percent of polyallylsucrose orpolyallylpentaerythritol, wherein the polymer is present at aconcentration of at least about 0.2 percent and the biologic solutioncomprises a physiologically acceptable electrolyte at a concentrationwhich is effective to lower substantially the viscosity imparted to thesolution by the polymer to a maximum of 2,000 cps.

, 2. A biologic according to claim 1, wherein the adjuvant is present ata concentration of about 0.2 to 0.5 percent.

3. A biologic according to claim 1, wherein the adjuvant and theelectrolyte are present at a concentration of about 0.25 percent andabout 0.7 percent, respectively.

4. A biologic according to claim 1, wherein the electrolyte is sodiumphosphate, sodium chloride, sodium citrate or magnesium sulfate.

5. A biologic according to claim 1, wherein the electrolyte is sodiumphosphate.

6. A biologic according to claim 5, wherein the adjuvant and theelectrolyte are present at a concentration of about 0.25 percent andabout 0.7 percent, respectively.

1. A BIOLOGIC SOLUTION COMPRISING AN ANTIGEN WHICH STIMULATES THEPRODUCTION OF ANTIBODIES AND AN ADJUVANT THEREFORE CONSISTINGESSENTIALLY OF A COLLOIDALLY WATER-SOLUBLE NEUTRALIZED POLYMER OFACRYLIC ACID CROSS-LINKED WITH FROM 0.75 TO 2.000 PERCENT OFPOLYALLYSUCROSE OR POLYALYLPENTAERYTHRITOL, WHEREIN THE POLYMER ISPRESENT AT A CONCENTRATION OF AT LEAST ABOUT 0.2 PERCENT AND THEBIOLOGIC SOLUTION COMPRISES A PHYSIOLOGICALLY ACCEPTABLE ELECTROLYTE ATA CONCENTRATION WHICH IS EFFECTIVE TO LOWER SUBSTANTIALLY THE VISCOSITYIMPARTED TO THE SOLUTION BY THE POLYMER TO A MAXIMUM OF 2,000 CPS.
 2. Abiologic according to claim 1, wherein the adjuvant is present at aconcentration of about 0.2 to 0.5 percent.
 3. A biologic according toclaim 1, wherein the adjuvant and the electrolyte are present at aconcentration of about 0.25 percent and about 0.7 percent, respectively.4. A biologic according to claim 1, wherein the electrolyte is sodiumphosphate, sodium chloride, sodium citrate or magnesium sulfate.
 5. Abiologic according to claim 1, wherein the electrolyte is sodiumphosphate.
 6. A biologic according to claim 5, wherein the adjuvant andthe electrolyte are present at a concentration of about 0.25 percent andabout 0.7 percent, respectively.